A. Bacteroides and Prevotella
Bacteroides is a genus of Gram negative, obligately anaerobic bacteria found in the gastrointestinal tracts of humans and animals. These bacteria function in metabolizing a wide range of carbohydrates. In humans, Bacteroides account for approximately 25% of the bacteria in the colon.
Prevotella ruminicola is a species of Gram negative, obligately anaerobic bacteria found in the rumen of cattle. P. ruminicola ferment carbohydrates such as hemicellulose, cellobiose, and starch and aid digestion and degradation of polysaccharides. P. ruminicola was previously classified as a member of the genus Bacteroides (Bacteroides ruminicola) because it has some characteristics associated with human colonic Bacteroides However, recent investigations showed that P. ruminicola shared less than 5% DNA-DNA homology with the colonic Bacteroides species. More detailed biochemical analyses also suggested that it belonged in a separate genus, Prevotella [See Shah, et al., Intl. J. Syst. Bacteriol., 40:205-208 (1990)].
Some progress has been made in connection with genetic manipulation of obligately anaerobic Bacteroides from the human colon. For example, shuttle vectors have been developed for use with some colonic Bacteroides which contain DNA from cryptic Bacteroides plasmids which are able to replicate in a number of different Bacteroides species [See Odelson, et al., Plasmid, 17:87-109 (1987); Salyers, et al., Crit. Rev. Microbiol., 14:49-71 (1987); Valentine, et al., J. Bacteriol.. 170:1319-1324 (1988)]. These vectors also contain sequences which allow them to replicate in E. coli and be mobilized out of E. coli by IncP plasmids. The IncP plasmids R751 and RP4 have been shown to mobilize DNA from E. coli to a variety of other species, including colonic Bacteroides species [See Salyers, et al., Crit. Rev. Microbiol., 14:49-71 (1987); Shoemaker, et al., J. Bacteriol., 166:959-965 (1986)]. One such E. coli-Bacteroides shuttle vector is pVAL-1 which contains cryptic Bacteroides plasmid pB8 -51 [Valentine, et al., J. Bacteriol., 170:1319-1324 (1988)].
Certain colonic Bacteroides strains have been found to harbor large self-transmissible elements carrying a tetracycline resistance ("Tc.sup.4 ") gene which are referred to as "conjugal elements" or "Tc.sup.r elements." Some of these Tc.sup.r elements also carry a clindamycin-erythromycin resistance ("Em.sup.r ") gene and are referred to as "Tc.sup.r Em.sup.r elements." These elements are not plasmids, but are integrated into the host chromosome.
The Tc.sup.r and Em.sup.r genes from a conjugal Tc.sup.r Em.sup.r strain of Bacteroides, Bacteroides thetaiotaomicron DOT, have been cloned, along with regions of the element that include transfer genes [Shoemaker, et al., J. Bacteriol., 171:1294-1302 (1989)]. The Tc.sup.r Em.sup.r element from B. thetaiotaomicron DOT has been designated "Tc.sup.r Em.sup.r -DOT."
These conjugal elements are able to transfer themselves from one colonic Bacteroides strain to another and to mobilize co-resident plasmids, not only from Bacteroides to Bacteroides, but also from Bacteroides to E. coli [See Odelson, et al., Plasmid, 17:87-109 (1987); Salyers, et al., Crit. Rev. Microbiol., 14:49-71 (1987); Thomson, et al., FEMS Microbiol. Letters, 61:101-104 (1989); Stevens, et al., J. Bacteriol. 172:4271-4279 (1990)]. Thus, the Tc.sup.r and Tc.sup.r Em.sup.r conjugal elements found in the colonic Bacteroides strains appear to be able to mediate mating pair formation between diverse genera of bacteria.
The conjugal element, Tc.sup.r Em.sup.r 12256, has been found to mobilize co-resident plasmids at high frequencies [See Valentine, et al., J. Bacteriol., 170:1319-1324 (1988)]. Furthermore, the Tc.sup.r Em.sup.r 12256 element appears to exhibit constitutive transfer, as opposed to other Tc.sup.r and Tc.sup.r Em.sup.r elements which require pre exposure to tetracycline to obtain maximum transfer frequencies.
Plasmid DNA has been introduced into some colonic Bacteroides using transformation techniques [See Salyers, et al., CRC Clinical Reviews in Microbiology, 14:49-71 (1987); Odelson, et al., Plasmid 17:87-109 (1987); Smith, J. Bacteriol., 164:294-301 (1985)]. For instance, one colonic Bacteroides species has been transformed by electroporation [Thomson, et al., FEMS Microbiol. Letters. 61:101-104 (1989)]. An E coli-colonic Bacteroides shuttle vector, pDP1, was isolated from Bacteroides uniformis and electroporated into B. uniformis at a frequency of 10.sup.6 transporants per microgram of DNA. However, the same plasmid, when isolated from E. coli EM24, gave only 10.sup.3 transporants per microgram of DNA.
Standard methods, however, appear to be inadequate in several respects for the transformation of the colonic Bacteroides. For example, large plasmids are difficult to introduce into these species by transformation techniques. Best results are obtained when the plasmid DNA is less that 5 kbp in size. Also, to obtain good rates of transformation, the donor plasmid must be isolated from the same strain used as the recipient. The difficulties encountered in crossing species lines are believed to be due to the presence of restriction barriers. Also, successful transformation of many species of colonic Bacteroides has been sporadic [See Odelson, et al., Plasmid, 17:102 (1987)]. Clearly, much improvement is needed in transformation methods for colonic Bacteroides.
Despite progress in understanding the genetics of colonic Bacteroides, P. ruminicola is not well understood genetically. There have been some biochemical studies of polysaccharide utilization by P. ruminicola, and a xylanase gene from P. ruminicola has been cloned and expressed in E. coli [See Whitehead, et al., Appl. Eviron. Microbiol., 55:893-896 (1989)].
Recently, a naturally-occurring plasmid carrying a gene coding for tetracycline resistance has been identified ("pRRI4") in P. ruminicola 223/M2/7. The pRRI4 plasmid was shown to transfer from P. ruminicola 223/M2/7 into P. ruminicola F101, but not into P. ruminicola 23, by conjugation [Flint, et al., Appl. Environ. Microbiol., 54:855-860 (1988)].
It has also been reported that the pRRI4 plasmid can be introduced into P. ruminicola F101 by electroporation, but not into P. ruminicola 118B, M384, GA33 by this method [Thomson and Flint, FEMS Microbiol. Letters, 61:101-104 (1989)]. This article also reports that pRRI4 isolated from P. ruminicola could not be introduced into B. uniformis, a colonic Bacteroides, by electroporation. Thomson and Flint also discloses that the E. coli-colonic Bacteroides shuttle vector pDPI could not be introduced into P. ruminicola by electroporation. This was true whether pDPI was extracted from B. uniformis or E. coli.
From the above discussion, it is clear that, prior to the present invention, the genetic manipulation of P. ruminicola was not possible. Little was known about the genetics of P. ruminicola, making the use of vectors that could be manipulated and amplified in a known host, such as E. coli, highly desirable However, no shuttle vectors were known that could be used in P. ruminicola. Transformation and conjugal transfer of pRRI4 was possible, but pRRI4 cannot be used as a shuttle vector due to its relatively large size (19.5 kbp) and its inability to replicate in E. coli.